One of the most important and dangerous types of foodborne infections which has been recognized recently is yersiniosis. This is caused by the bacteria Yersinia enterocolitica. The genus Yersinia is widespread in the environment and can grow at refrigeration temperatures to cause problems in chilled food products stored for extended periods of time. There is concern from medical authorities that the food supply ought to be monitored for the presence of this microorganism, although the food industry is not yet routinely monitoring the presence of Yersinia in products. However, many authorities expect that Yersinia will become as important as Listeria is now.
The genus Yersinia is composed of a collection of Gram-negative, facultative anaerobic bacilli that share with other genera of the family Enterobacteriaceae a number of common morphologic, biochemical and serologic features. At present 11 species are recognized within this genus, three of which (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) have been shown unquestionably to be human pathogens (1,2). Yersinia are known to inhabit a wide range of animal and environmental sources, with some species displaying a host-specific tropism for colonization and/or infection. An interesting characteristic of most Yersinia spp. is their temperature-dependent expression of a number of phenotypic traits such as motility, biochemical properties and virulence-associated markers (3).
Y. enterocolitica is the most common agent of this genus recovered from patients and is usually associated with sporadic cases of gastroenteritis and mesenteric lymphadenitis. In the past two decades there has been a dramatic increase in the frequency of the isolation of this organism from both clinical and nonclinical specimens (4). In several countries, including the Netherlands, Belgium, Canada, and Australia, Y. enterocolitica has surpassed Shigella and rivals Salmonella and Campylobacter as a cause of acute bacterial gastroenteritis (5,6). Extra-intestinal illnesses attributed to Y. enterocolitica have been reported and include bacteraemia, arthritis, pharyngitis and pyomyositis (1,3). There are over 50 serotypes in this species; however, only five, designated 0:3, 0:5, 27; 0:6,30; 0:8 and 0:9, are generally regarded as pathogenic for humans (7).
Among the pathogenic serotypes, 0:3 and 0:9 have been commonly associated with Y. enterocolitica strains recovered from Europe, Scandinavia and Canada. Previously in the United States, serotypes 0:8 and 0:5, 27 were the predominating serotypes. However, Bottone (8) and Shayegani et al. (9) recently reported on the emergence of serotypes 0:3 in New York City and State, indicating changes in the U.S. pattern.
Yersinia has often been described as a food-borne pathogen (10,11), a deduction supported by descriptions of outbreaks in which some common vehicle was likely (12,13) and by the fact that a food animal, swine, is a reservoir of the common pathogenic serotype 0:3.
Geographic differences in the frequency and distribution of Y. enterocolitica infection are apparent. In Europe, Sporadic infections caused by serotypes 0:3 and 0:9 are common (5,14), but outbreaks of disease are rare. In the United States, sporadic disease is associated with multiple serotypes and is relatively uncommon (15,16), but five outbreaks have been caused by serotype 0:8. Transmission of this pathogenic bacteria to man appears to be mainly via the digestive tract, and may include the ingestion of contaminated food, contact with an infected animal, and person-to-person transmission through hand-to-mouth contact (17).
Thus, Y. enterocolitica has recently emerged as an important and dangerous foodborne pathogenic bacteria, the causative agent of yersiniosis, which is capable of growth at refrigeration temperature in various foods.
Y. enterocolitica infection of humans is due either to strains circulating from person to person or to strains found in foods or in other environmental sources that infect humans by the oral route (17).
One of the major problems in identifying the vehicle of transmission for yersiniosis outbreaks and identification of Y. enterocolitica as the causative agent of infection is the lack of appropriate isolation media.
The inability to identify the vehicle of transmission for yersiniosis outbreaks and delayed recognition of Y. enterocolitica is mainly attributed to the lack of appropriate isolation media and techniques with consequent lack of awareness of its presence in clinical specimens and foods (18). Y. enterocolitica strains show a marked degree of variability in their ability to grow on routinely-used enteric media, hence, those media are inadequate for primary isolation (3,19). Laboratory methods first used for recovery of Y. enterocolitica from foods were based primarily on cold enrichment techniques previously described for examination of faeces (20,21) and there have been few improvements since. Isolation of Y. enterocolitica from food is more difficult than from faeces taken from patients with active infections in that the number of Yersinia may be smaller, and the background flora is likely to be greater in both number and variety. Pai et al. (22) have concluded that cold enrichment is not essential for recovery of Y. enterocolitica from stool specimens taken from patients with symptoms and VanNoyan et al. (23) suggested that the additional recoveries obtained by cold enrichment were types of Y. enterocolitica that were not clinically important. In the case of foods, however, enrichment provides the only certain way for selecting out low number of Yersinia from the total microbial population.
Phosphate-buffered saline (PBS) has been a common cold-enrichment medium for both faeces and foods, Vanpee and Stragier (24) suggested that a rich broth such as trypticase soy broth is preferred for cold-enrichment.
Kounev (25) reported that phosphate-buffered saline (pH 7.6) was the best medium for the recovery of heat-injured cells of Y. enterocolitica serotype 0:3 from cooked sausage when incubated at 25.degree. C. for 24-48 h. Incubation at 4.degree. C. appeared to present an additional stress factor and more nutritive media interfered with the isolation of Y. enterocolitica.
Due to the long time periods required for cold-enrichment, there have been various efforts to devise selective enrichments which could be incubated for shorter times at higher temperatures. Inone and Kurose (26) used selenite medium with Novobiocin at 37.degree. C. with less success than cold-enrichment. Lee et al. (27) described two modified selenite media incubated at 22.degree. C. that were effective for recovery of certain strains of Y. enterocolitica.
There have been only a few attempts to devise two-step enrichment techniques for recovery of Y. enterocolitica from raw milk. Vidon and Delmas (28) also obtained greater recovery from raw milk by using selective enrichment of their own formulation incubated at 28.degree. C. for 48 hours after cold enrichment for 1 month. A selenite-malachite green ticarcillin enrichment without added nutrient was recommended by Lee et al. (29), especially for the recovery of the more fastidious strains from meat. Schiemann (18) described a two-step procedure, involving pre-enrichment in yeast-extract rose bengal (YER) broth, followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for the isolation of Y. enterocolitica from foods.
The major disadvantages of BOS medium are:
(a) The complexity of its manufacture and that it is not yet commercially available; and PA1 (b) there is no evidence to show that this medium can recover the heat-and cold-injured cells of Y. enterocolitica. PA1 (a) a proteolytic digest of protein; PA1 (b) an extract selected from a yeast extract and a beef extract; PA1 (c) an alkali metal chloride or alkaline earth metal chloride; . PA1 (d) bile salts, sodium deoxycholate or sodium cholate; and PA1 (e) 5-chloro-2-(2,4-dichlorophenoxy) phenol. PA1 20 g/L POLYPEPTONE.TM. peptone PA1 2 g/L yeast extract PA1 2 g/L sodium chloride PA1 0.002 g/L triclosan PA1 1 g/L sodium deoxycholate PA1 10 g/L disodium hydrogensphosphate PA1 2 g/L sodium oxalate PA1 2 g/L pyruvic acid PA1 0.5 g/l glycine PA1 0.01 g/L magnesium sulphate PA1 0.005 g/L ferrous ammonium sulphate PA1 10 g/L sucrose PA1 0.005 g/L cefsulodin; said solution having a pH selected from 7.2 to 7.9. PA1 (a) a peptone; PA1 (b) an extract selected from a yeast extract and a beef extract; PA1 (c) an alkali metal chloride or alkaline earth metal chloride; PA1 (d) bile salts or sodium deoxycholate or sodium cholate; and PA1 (e) triclosan. PA1 (a) incubating said product in an aqueous growth medium having a composition comprising an effective amount of trypticase soy broth, yeast extract, magnesium sulphate, pyruvic acid and sucrose, at a pH selected from 7.2 to 7.9 to provide an enriched solution of Y. enterocolitica; and PA1 (b) incubating said enriched solution in an aqueous Y. enterocolitica growth-enhancing composition comprising an effective amount of an aqueous solution as hereinabove defined.
Several other methods or modification of the existing methods have been proposed but are less widely used. Wauters et al. (30) have recommended a new enrichment methodology for the recovery of the most pathogenic serotype (0:3) from meat by using three selective agents, triclosan, ticarcillin and potassium chlorate, but this study does not explain whether the selective supplements are inhibiting other background microorganisms or enhancing the growth of Y. enterocolitica. Also the inhibitory action of these selective supplements are not shown against other background flora in this study.
A new three-step procedure (TSP) for the recovery of Y. enterocolitica 0:3 from frozen meat, salted and dried, meat products and cooked perishable sausages has been developed by Kouner (31). The TSP is based mainly on enrichment in 0.15M phosphate-buffered saline at 25.degree. C.
The media and method used in the above studies are applicable only for the recovery of Y. enterocolitica serotype 0:3. No information is available for the efficiency of the above media and methods for the recovery of Y. enterocolitica serotypes other than 0:3.
Landgraf et al. (32) recommended an improved enrichment procedure for recovery of Y. enterocolitica from milk by modification of trypticase soy broth (TSB) with addition of polymyxin B and novobiocin. Not enough evidence has been published to show that this medium has inhibitory action against other Gram-negative bacteria or enhances the growth of all serotypes of Y. enterocolitica. This procedure and medium has been studied for the recovery of Y. enterocolitica only from milk and the author has also indicated the difficulties in the recovery of Y. enterocolitica from the raw milk because of the presence of other background microorganisms. All the media and procedures mentioned above have the disadvantages of either a long incubation time or complexity of preparation. As a result, none of the above media are commercially available.
The disadvantages of other techniques such as polymerase chain reaction (PCR) and DNA probes are that these techniques cannot detect low initial cell numbers in the sample or differentiate between the dead and live cells. Due to the high degree of similarities between the genetic material of most Gram-negative bacteria and being very much prone to contamination, the use of these techniques may lead to a false positive reaction.
Thus to date, no simple, selective, enrichment broth medium is available which is efficient for the isolation and recovery of all serotypes of Y. enterocolitica from environmental and clinical samples.